Hematopoietic stem cells (HSCs) represent one of the most flexible and well-studied tissue stem cells in mammals, and are clinically important for the treatment of hematologic malignancies, congenital bone marrow syndromes, and other malignancies. Our understanding of the molecular pathways that underlie the self-renewal versus differentiation decisions is insufficient to allow us to safely manipulate HSCs for clinical benefit. Our preliminary studies and published work demonstrates that the Mixed Lineage Leukemia (MLL) protein is essential for sustaining HSCs during development and homeostasis. We have developed and utilized conditional gene ablation approaches to define a window of development in which HSCs become dependent on MLL for their expansion. This window coincides with the establishment of hematopoiesis in the bone marrow. We have also identified a small, unique network of genes in adult HSCs that we hypothesize are direct transcriptional targets of MLL and are important effectors of its HSC maintenance function. Our overall objective is to define the genetic network within which MLL operates in fetal and adult HSCs, since key features of HSC function, proliferation and self-renewal are distinct between these two developmental periods. To achieve this objective, we will: 1) identify MLL-dependent genes in murine fetal HSCs and compare these to our adult HSC data, 2) determine the pattern of MLL binding in fetal HSCs using human HSC-enriched populations, 3) determine the niche and developmental stage defining the beginning of MLL dependent HSC expansion/homeostasis, and 4) define the MLL-dependent transcriptional network and mechanisms that operate in adult murine HSCs to promote self-renewal. Accomplishing these aims will result in important new insights into a critical but understudied pathway that regulates HSC function and self-renewal. By using the best features of human and mouse model systems, and combining state-of-the-art molecular and genomic techniques with rigorous developmental studies, we hope to discover safe and effective means to manipulate HSCs for clinical benefit. PUBLIC HEALTH RELEVANCE: The hematopoietic or blood-forming system must continuously regenerate all blood cell types throughout the lifetime of adult mammals. This regeneration is ultimately fueled by a small and unique population of cells termed hematopoietic stem cells (HSCs). This cell type underlies the success of bone marrow transplantation, which is a treatment for leukemia and other blood disorders. We propose to investigate the molecular pathways that are regulated by the Mixed Lineage Leukemia (MLL) gene, because this gene is essential for sustaining HSCs and the entire hematopoietic system in mice and, we hypothesize, in humans. We will determine the molecular pathways controlled by MLL in adult and fetal hematopoietic stem cells using state-of- the art genomic approaches coupled with genetically engineered animal models. By understanding how these pathways are regulated, we will reveal fundamental new insights into methods to expand HSCs and insights into the mechanisms by which leukemia is initiated.